Direct hapten-linked competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of O-pinacolyl methylphosphonic acid

Abstract

Immunoassay detection of O-pinacolyl methylphosphonic acid (PMPA) employing direct coating of N-2-aminoethyl-O-pinacolyl methylphosphonate (hapten B) on microtiter plates is reported. Coating was achieved by covalently linking hapten B to a glutaraldehyde (GA) polymer network directly bound to the polystyrene (PS) surface of a standard 96-well microtiter plate. 4-(2-(O-Pinacolylmethylphosphoryl amino)ethyl amino)-4-oxobutanoic acid (hapten A)–ovalbumin (OVA) conjugate served as the coating antigen for comparison with direct hapten B-coated plates in the CIELISA format. The developed assay employing direct hapten B coated plates demonstrated enhanced sensitivity with the IC₅₀ value for PMPA being 0.027 μg mL⁻¹. The assay could detect PMPA even at the concentration of 0.006 μg mL⁻¹. The mean recovery of standard PMPA (spiked in water) was found to be 83.7%.